Towards pharmacokinetic boosting of phenoxymethylpenicillin (penicillin-V) using probenecid for the treatment of bacterial infections

In the face of increasing antimicrobial tolerance and resistance there is a global obligation to optimise oral antimicrobial dosing strategies including narrow spectrum penicillins, such as penicillin-V. We conducted a randomised, crossover study in healthy volunteers to characterise the influence of probenecid on penicillin-V pharmacokinetics and estimate the pharmacodynamics against Streptococcus pneumoniae. Twenty participants took six doses of penicillin-V (250 mg, 500 mg or 750 mg four times daily) with and without probenecid. Total and free concentrations of penicillin-V and probenecid were measured at two timepoints. A pharmacokinetic model was developed, and the probability of target attainment (PTA) calculated. The mean difference (95% CI) between penicillin-V alone and in combination with probenecid for serum total and free penicillin-V concentrations was significantly different at both timepoints (total: 45 min 4.32 (3.20–5.32) mg/L p < 0.001, 180 min 2.2 (1.58–3.25) mg/L p < 0.001; free: 45 min 1.15 (0.88–1.42) mg/L p < 0.001, 180 min 0.5 (0.35–0.76) mg/L p < 0.001). There was no difference between the timepoints in probenecid concentrations. PTA analysis shows probenecid allows a fourfold increase in MIC cover. Addition of probenecid was safe and well tolerated. The data support further research into improved dosing structures for complex outpatient therapy and might also be used to address penicillin supply shortages.


INTRODUCTION
1.1 BACKGROUND Probenecid, p-(di-n-propylsulfamyl)-benzoic acid, was developed in 1949 with the purpose of decreasing the renal clearance of penicillin. 1 The development of probenecid stemmed from the requirement to ration supplies of penicillin during World War II.Its mechanism of action is thought to be to the competitive inhibition of organic anion transporters, which are responsible for the excretion of organic agents, such as penicillin. 2 Reduction in renal clearance of penicillin with probenecid demonstrated significant increases in serum concentrations, meaning that lower doses of drug were required for similar pharmacokineticpharmacodynamic (PK-PD) target attainment.Probenecid's influence on penicillin clearance became mainly academic in the post-war era as our capability to produce more diverse, cheaper, and safer beta-lactam antibiotics rapidly expanded. 1Probenecid remains a recommended adjunct in the management of some sexually transmitted diseases to support therapeutic target attainment in compartments, such as cerebrospinal fluid. 3However, its potential important broader role in preserving the effectiveness of beta-lactams through the optimisation of beta-lactam PK needs to be considered.
The World Health Organisation (WHO) AWaRe criteria require narrow spectrum antimicrobials, such as the penicillins, to be available in appropriate type, dose, and duration to treat common infections. 4With increasing drug-resistance within common causative organisms, such as in streptococcal infections, new methods to optimise the delivery of Access agents and protect the use of broader Watch and Reserve antimicrobials is required. 4It is not always possible to simply prescribe higher doses of an antibiotic to overcome problems such as increasing drug-resistance.In some instances, oral drug absorption or gastrointestinal side effects associated with high doses limit escalation of therapy.Some agents are not licenced for use at doses required to obtain acceptable PK-PD targets for high minimum-inhibitory concentration (MIC) organisms.Some settings, such as outpatient parenteral antibiotic therapy (OPAT) do not allow for multiple daily dosing schedules of an intravenous drug, meaning that an oral or once daily alternative intravenous option is required.Probenecid offers a means of optimising the delivery of oral and intravenous antimicrobial therapy through manipulation of beta-lactam PK without significant changes to antimicrobial dosing.

RATIONALE FOR CURRENT STUDY
This study aims to build on previous work characterising the PK of penicillin-V to explore the potential impact of probenecid on PK-PD target attainment.Achievement of the aims of this study would provide data to support the design of experimental studies exploring the clinical impact of probenecid on treatment outcomes and also provide a rationale for exploration of probenecid's effects on a larger number of beta-lactam antibiotics.
Hypothesis: Addition of probenecid to oral phenoxymethylpenicillin (penicillin-V) has a clinically relevant effect on pharmacokinetic-pharmacodynamic (PK-PD) target attainment.

Statistical analysis:
PK modelling of data will be undertaken using Pmetrics in R. Non-linear mixed effects modelling will be used to estimate the posterior estimates for individual PK.Probenecid exposures influence on clearance will be explored as a covariate in the model.
Probability of target attainment (40% fT>MIC) for common organisms (e.g.streptococci) will be estimated.Descriptive statistics will be applied to the estimated population parameters and simulated PTAs to facilitate comparison of the data.

Summary of method:
Participants will be screened and consented to attend Imperial College Clinical Research Facility (CRF) at Hammersmith Hospital on two study visits, at least 7 days apart.For one visit (randomised), participants will be required to take penicillin-V only.For their other visit, they will take penicillin-V plus probenecid at standard recommended dose.Prior to the study visits, participants may be required to have taken 36-hours of penicillin +/probenecid, documenting this in a dosing diary.On arrival at the CRF, the participant will take an observed dose of penicillin +/-probenecid.They will undergo blood draw via needle phlebotomy or a cannula (participant choice) at 45 and 180 minutes post the observed.Samples will be spun down and frozen at -80 o C.They will subsequently be analysed using an in-house HPLC-MS/MS methodology to determine total and free-unbound drug concentration.
For analysis, data from this study will be pooled with rich PK data from a prior study that assessed plasma concertation of penicillin-V in healthy volunteers.Pmetrics in R will be used to model the data looking to explore the effect of probenecid on clearance of free-penicillin-V.Probability of target attainment for streptococci species will also be estimated to evaluate the potential clinical impact of the addition of probenecid to routine penicillin-V use.Rich PK data for intravenous benzylpenicillin will be used to estimate PK-PD target attainment and PTAs for intravenous formulations, allowing direct comparison of oral and IV regimes.

Medication used in the study:
Penicillin-V and probenecid used in this study will be prescribed by a qualified doctor.It will be supplied by Imperial College Healthcare NHS Trust pharmacy.
Probenecid will be dosed at 500mg four-time a day (QDS) for all participants.Note: probenecid is not licenced for use in the United Kingdom.It is licenced in the EU.The dose selected is based on the probenecid summary of product characteristics, known doses used safely in the treatment of neuro-syphilis with benzylpenicillin as part of British STI treatment guidance, and is in line with published literature and guidelines for unlicensed use in the treatment of infection within UK and international organisations.Probenecid will be sourced from BioPhausia AB, Sweeden via Imperial College Healthcare NHS Trust Pharmacy.Dosing will be in line with their SmPC.
Penicillin-V within the study will be dosed as follows for participants: 750mg QDS penicillin-V Dose escalation in increments of 250mg has been selected to provide a wide safety margin even at higher doses.Probenecid is estimated to reduce penicillin clearance by approximately 30%.Given that the maximum licenced dose of penicillin-V in the UK is 4g / day, we estimate that for a 30% reduction in clearance, 750mg QDS should remain within this margin of safety.Furthermore, given that doses of intravenous penicillin achieve much greater concentrations with limited side effects, we do not anticipate issues with toxicity within this study.
Both drugs will be dispensed by Imperial College Hospital NHS Trust pharmacy with labelling performed in-house in alignment with Annex 13 under the Reg 37 hospital exemption.

STUDY OUTCOME MEASURES
Primary outcome measure: 1. Total and unbound penicillin-V concentrations at 45 and 180 minutes post observed penicillin-V dose with and without probenecid.
Secondary outcome: 1. Probability of target attainment for common infections treated with penicillin (e.g.Streptococcus spp.) will be estimated.2. PK-PD model describing the influence of probenecid on penicillin-V in healthy volunteers.

PRE-RANDOMISATION EVALUATIONS
Pre-registration evaluation will include: -Clinical assessment -Confirmation of prior penicillin use and no documented penicillin allergy.
-Pregnancy test in female participants.
-Previously taken penicillin-based antibiotics without allergic response.

EXCLUSION CRITERIA
-Lacking capacity to consent.
-Pregnant or likely to become pregnant during study period.
-Symptoms consistent with active infection.
-History of gout or uric acid kidney stones.
-Taking regular medication that may interact with probenecid including, but not limited to methotrexate, lorazepam, acetaminophen, oral hypoglycaemic medication, sulfa containing drugs, non-steroidal anti-inflammatory drugs.
-History of evidence of any medical, neurological, or psychological condition that would expose the subject to an undue risk of a significant adverse event or interfere with study assessments during the course of the trial as determined by the clinical judgement of the investigator.
-Recent involvement in other research (within prior 3 months).

WITHDRAWAL CRITERIA
Participants can withdraw at any point from the study without providing a reason for their decision.If they choose to withdraw after entry into the study any samples provided to this point (with their consent) will be retained for use in the study.
If a participant develops any exclusion criteria after enrolment, they will be withdrawn from the study immediately.

Study stopping rules:
If information were to arise during the course of this study that would negate the results or suggest any safety concerns, the study will be stopped immediately with participants withdrawn and an update provided to them explaining the rationale for this.
IMP treatment will be discontinued individually if a study participant presents a severe or serious adverse event considered related to the study IMPs.
The trial will be halted if any of the following is observed: • A serious adverse reaction (i.e. a serious adverse event considered at least possibly related to the IMP administration) in one subject.• Severe adverse reactions (i.e.severe adverse events considered as, at least, possibly related to the IMP administration) in two subjects, independent of within or not within the same system-organ-class.
If the trial is halted due to any of the study stopping rules detailed above, the trial will only be re-started after regulatory authority approval via a substantial amendment.

RANDOMISATION OR REGISTRATION PRACTICALITIES
Pre-randomisation screening will require clinical assessment, checking of eligibility, pregnancy testing if female, and screening blood tests (FBC, LFT, Renal Profile, HIV, HBV, & HCV).
Randomisation will be performed using simple randomisation.This will aim for 50% of individuals to commence on intervention and 50% comparator arm of the study.A randomisation list will be generated prior to commencement of the study.Participants will then be added sequentially onto this list as they enter the study.
This study will be unblinded.

CONTRACEPTION REQUIREMENTS FOR FEMALE PARTICIPANTS
Probenecid crosses the placental barrier and appears in cord blood, and the use of this drug in patients that are women of childbearing potential requires that the anticipated benefit be weighed against possible hazards.As this clinical trial is in healthy volunteers whom are not deriving any benefit by taking part in the trial, female participants must follow adequate contraception advice during the full participation in the trial.
This trial follows the contraception guidance for female participants set out in the Clinical Trial Facilitation Group (CTFG) guidance on 'Recommendations related to contraception and pregnancy testing in clinical trials'.
A woman of childbearing potential (WOCBP) is defined as a "female participant that is fertile, following menarche and until becoming post-menopausal unless permanently sterile.Permanent sterilisation methods include hysterectomy, bilateral salpingectomy and bilateral oophorectomy.A postmenopausal state is defined as no menses for 12 months without an alternative medical cause.A high follicle stimulating hormone (FSH) level in the postmenopausal range may be used to confirm a postmenopausal state in women not using hormonal contraception or hormonal replacement therapy (HRT).However, in the absence of 12 months of amenorrhea, confirmation with more than one FSH measurement is required".
For WOCBP, acceptable methods of contraception must be used for the total duration of enrolment in the trial.Acceptable methods of contraception to be included within this study are defined within section 4 of the CTFG guidance as: Acceptable and highly effective methods of birth control • Combined hormonal contraception (oestrogen and progesterone containing) associated with inhibition of ovulation (oral, intravaginal, or transdermal).• Progesterone only hormonal contraception associated with the inhibition of ovulation (oral, injectable, or implantable).
• Sexual abstinence* *NB: sexual abstinence is considered a highly effective method only if defined as refraining from heterosexual intercourse during the entire period of risk associated with the study treatments.The reliability of sexual abstinence needs to be evaluated in relation to the duration of the clinical trial (estimated at 48 hours for each dosing interval) and the preferred and usual lifestyle of the subject.Clear documentation of this decision must be made at the time of screening.

Acceptable but not considered as highly effective methods of birth control
• Progesterone-only oral hormonal contraception, where inhibition of ovulation is not the primary mode of action.• Male or female condom with or without spermicide.
• Double barrier methods (e.g.male condom with cap, diaphragm, or sponge with spermicide) are also considered as acceptable, but not highly effective methods of birth control.

TREATMENTS
6.1 TREATMENT ARMS All treatments (penicillin-V and probenecid) used within this study will be supplied by Imperial College Healthcare NHS Trust pharmacy.
Probenecid will be dosed at 500mg QDS for all participants.It will be supplied by Imperial College Healthcare NHS Trust pharmacy.The formulation will be tablet form (500mg tablets).
Penicillin-V will be dosed as follows for participants: Group 1 (n=24) 500mg QDS penicillin-V Group 2 (n=13) 250mg QDS penicillin-V Group 3 (n=13) 750mg QDS penicillin-V It will be supplied by Imperial College Healthcare NHS Trust pharmacy.The formulation will be tablet form (250mg tablets).All participants will be required to commence treatment 36 hours prior to the CRF visit.They will take a maximum of 6 doses of penicillin-V +/-probenecid prior to attending the CRF.They will then take a further observed dose of penicillin-V +/-probenecid during the CRF visit (at time-point 0 minutes).The requirement for 6 doses prior to attending the CRF visit is to ensure that drug concentration (penicillin-V +/-probenecid) is at steady state (normally requires approximately 5 doses) prior to drug level sampling.In total, throughout the study (both visits), participants will take a maximum of 14 doses of penicillin-V and 7 doses of probenecid.This treatment interval has been selected as it is the shortest feasible duration to ensure patients arrive for the study visit at steady-state, whilst minimising the potential risks to safety in terms of drug interactions and adverse events.

DOSE MODIFICATIONS FOR TOXICITY
No dose reductions are anticipated in this study.Participants will be required to have normal renal function.The dose of penicillin-V is within its licensed range.The dose of probenecid used is widely used in clinical practice with minimal reported side effects.Dose escalation in increments of 250mg has been selected to provide a wide safety margin even at higher doses.Probenecid is estimated to reduce penicillin clearance by approximately 30%.Given that the maximum licenced dose of penicillin-V in the UK is 4g / day, we estimate that for a 30% reduction in clearance, 750mg QDS should remain within this margin of safety.Furthermore, given that doses of intravenous penicillin achieve much greater concentrations, we do not anticipate issues with toxicity within this study.

6.3
PREMEDICATION No premedication will be required as part of this study.

INTERACTION WITH OTHER DRUGS
Participants will be informed of potential interactions of probenecid and other drugs.These include but a not limited to: methotrexate, lorazepam, acetaminophen, oral hypoglycaemic medication, sulfa containing drugs, non-steroidal anti-inflammatory drugs.
Participants will be asked not to take other medications if possible during the dosing visits.If they require paracetamol, they will be advised to take 500mg QDS maximum.

DISPENSING AND ACCOUNTABILITY
All medications used within this study will be supplied via the NHS suppliers which will be overseen by Imperial College Healthcare NHS Trust Clinical Trials Pharmacy team.The investigation medicinal product (IMP) will be used in line with the summary of medicinal product characteristics obtained from the EU-licensed supplier.All medications will be prescribed by a physician within the research team at the doses required per participant.IMP 1: Probenecid (tablet form) -EU market authorisation BioPhausia AB (MA number: 4522) IMP 2: Penicillin-V (tablet form) -UK market authorisation Sandoz BmB (MA number: PL 04520/0005)

SAFETY MONITORING ASSESSMENTS
If there are concerns with potential participant safety as defined by investigator criteria or clinical indication at any time during the study a safety monitoring assessment will be performed by a clinician within the study team.This may include physical examination, blood testing, and measurement of participant vital signs.

7.
Pharmacovigilance 7.1 DEFINITIONS Adverse Event (AE): any untoward medical occurrence in a patient or clinical trial subject administered a medicinal product and which does not necessarily have a causal relationship with this treatment.An AE can therefore be any unfavourable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of an investigational medicinal product (IMP), whether or not considered related to the IMP.
Based on the investigator judgement, an AE will be defined as: Mild: An event that does not affect the activities of daily living.It is easily tolerated.The effect is mild and does not impact the individual.
Moderate: Affects normal activities of daily living.Affects the ability of one to carry out tasks which they normally do.It causes discomfort and distress to the individual.
Severe: An event that prevents normal daily activities.
Adverse Reaction (AR): all untoward and unintended responses to an IMP related to any dose administered.All AEs judged by either the reporting investigator or the sponsor as having reasonable causal relationship to a medicinal product qualify as adverse reactions.The expression reasonable causal relationship means to convey in general that there is evidence or argument to suggest a causal relationship.
Unexpected Adverse Reaction: an AR, the nature or severity of which is not listed in the reference safety information (RSI) e.g.list of expected medical events within investigator's brochure for an unapproved investigational product or section 4.8 of the summary of product characteristics (SmPC) for an authorised product.When the outcome occurs this adverse reaction should be considered as unexpected.Side effects documented in the SmPC which occur in a more severe form than anticipated are also considered to be unexpected.

Serious Adverse Event (SAE)
or Serious Adverse Reaction: any untoward medical occurrence or effect that at any dose: • Results in death.
• Is life-threatening -refers to an event in which the subject was at risk of death at the time of the event; it does not refer to an event which hypothetically might have caused death if it were more severe.• Requires hospitalisation, or prolongation of existing inpatients' hospitalisation.
• Results in persistent or significant disability or incapacity.
• Is a congenital anomaly or birth defect.
Medical judgement should be exercised in deciding whether an AE/AR is serious in other situations.Important AE/ARs that are not immediately life-threatening or do not result in death or hospitalisation but may jeopardise the subject or may require intervention to prevent one of the other outcomes listed in the definition above, should also be considered serious.
Suspected Unexpected Serious Adverse Reaction (SUSAR): any suspected adverse reaction related to an IMP that is both unexpected and serious.

CAUSALITY
Most adverse events and adverse drug reactions that occur in this study, whether they are serious or not, will be expected treatment-related toxicities due to the drugs used in this study.The assignment of the causality should be made by the investigator responsible for the care of the participant using the definitions in the table below.
If any doubt about the causality exists the local investigator should inform the study coordination centre who will notify the Chief Investigators.The pharmaceutical companies and/or other clinicians may be asked to advise in some cases.
In the case of discrepant views on causality between the investigator and others, all parties will discuss the case.In the event that no agreement is made, the MHRA will be informed of both points of view.

Relationship Description Unrelated
There is no evidence of any causal relationship Unlikely There is little evidence to suggest there is a causal relationship (e.g. the event did not occur within a reasonable time after administration of the trial medication).There is another reasonable explanation for the event (e.g. the participant's clinical condition, other concomitant treatment).

Possible
There is some evidence to suggest a causal relationship (e.g. because the event occurs within a reasonable time after administration of the trial medication).However, the influence of other factors may have contributed to the event (e.g. the participant's clinical condition, other concomitant treatments).

Probable
There is evidence to suggest a causal relationship and the influence of other factors is unlikely.

Definitely
There is clear evidence to suggest a causal relationship and other possible contributing factors can be ruled out.

Not assessable
There is insufficient or incomplete evidence to make a clinical judgement of the causal relationship.

REPORTING PROCEDURES
All adverse events should be reported.Depending on the nature of the event the reporting procedures below should be followed.Any questions concerning adverse event reporting should be directed to the study coordination centre in the first instance.A flowchart is given below to aid in the reporting procedures.All AE and SAEs must be recorded from the time of consent, until the end of the last study visit.

Non serious AR/AEs
All such toxicities, whether expected or not, should be recorded in the toxicity section of the relevant case report form and sent to the study coordination centre within one month of the form being due.

Serious AR/AEs
Fatal or life threatening SAEs and SUSARs should be reported on the day that the local site is aware of the event.The SAE form asks for nature of event, date of onset, severity, corrective therapies given, outcome and causality (i.e.unrelated, unlikely, possible, probably, definitely).The responsible investigator should sign the causality of the event.Additional information should be sent within 5 days if the reaction has not resolved at the time of reporting.

Figure .
Figure.Total plasm penicillin concentration -time profiles for healthy volunteers receiving oral penicillin-V.